An aspirin derivative, a preparation method and application thereof in preventing cardiovascular and cerebrovascular diseases

Abstract

The application discloses an aspirin derivative, a preparation method thereof and application of the aspirin derivative in prevention of cardiovascular and cerebrovascular diseases. A molecular structure expression of the aspirin derivative is as follows. The preparation method of the aspirin derivative comprises the following steps: S1, oxalyl chloride is added into an aspirin solution and stirred, dimethylformamide is further added as a cosolvent, stirring is carried out at room temperature for 5-10 hours, and 2-(acetyloxy)benzoyl chloride is generated through reaction; S2, saturated sodium bicarbonate solution is added into a histidine proline solution, the mixture is treated through ice bath, 2-(acetyloxy)benzoyl chloride is further added, and stirring reaction is carried out for 10-24 hours; the obtained reaction solution is poured into a hydrochloric acid solution, a precipitate is obtained through filtration, and the aspirin derivative is prepared through extraction and purification.

Description

TECHNICAL FIELD

[0001] The present application relates to an aspirin derivative, in particular to an aspirin derivative, a preparation method and the application thereof in preventing cardiovascular and cerebrovascular diseases. BACKGROUND

[0002] At present, aspirin has been widely used in the secondary prevention of cardiovascular and cerebrovascular diseases, and is considered as an indispensable preventive measure for the treatment of acute coronary syndrome and acute ischemic stroke. Aspirin not only has the effect of anti-platelet aggregation, which can prevent the formation of thrombosis and embolism related to atherosclerosis (AS) lesions, but also has the cardiovascular protection effect of anti-inflammation and stabilization of plaques, anti-oxidation, protection of vascular endothelial cells and inhibition of smooth muscle cell proliferation and vasodilation, which are important pharmacological mechanisms of aspirin in effectively preventing cardiovascular and cerebrovascular events.

[0003] As a secondary prevention drug, aspirin cannot treat diseases from the root, and needs to be taken for a long time, but it has a great stimulation on the gastrointestinal tract and can cause side effects such as gastrointestinal bleeding. In order to overcome the damage of aspirin to the gastric mucosa, some people have made aspirin into enteric-coated tablets, which can avoid the direct stimulation of aspirin on the gastric mucosa, but cannot solve the stimulation of aspirin on the intestinal mucosa. Therefore, it is urgent to develop a new substitute drug that can reduce the gastrointestinal side effects of aspirin.

[0004] Patent CN104721800B proposes an aspirin composition, which combines aspirin with traditional Chinese medicine raw materials to obtain a Chinese and western medicine with good synergistic effect and small side effects. However, the effects of traditional Chinese medicine and western medicine are different, and the combination of drugs can easily change the properties of the drugs and reduce the therapeutic effect of the drugs, and the quality of traditional Chinese medicine is difficult to control, so there is a long research process before the scheme can be applied in clinical practice.

[0005] Patent CN104276962B designs acetylsalicylic acid diethylaminoethyl sulfate as a substitute for aspirin, which greatly increases the solubility of the drug through water-soluble-oil-soluble balance structure, so that transdermal absorption is possible, which is expected to reduce the stimulation of the gastrointestinal tract. However, from the test data of Example 3, it can be seen that it is decomposed into salicylic acid in vivo, not aspirin. It is known in the art that aspirin is not only a prodrug of salicylic acid, but also a drug with its own specific action mode. Without disclosing the specific therapeutic effect of the derivative, it is impossible to determine whether it can achieve the same therapeutic effect as aspirin in preventing cardiovascular and cerebrovascular diseases. SUMMARY

[0006] To solve the above technical problems, the present application first proposes an aspirin derivative. The aspirin derivative has the structural groups of aspirin and histidine proline at the same time, and the aspirin is modified by the histidine proline dipeptide structure. It is found in research that the dipeptide can enhance the prevention and treatment effect of aspirin on cardiovascular and cerebrovascular diseases, and has almost zero toxicity to the body, and can improve the side effects caused by taking aspirin.

[0007] Based on the second aspect of the present application, a preparation method of the aspirin derivative is also provided. The aspirin derivative can be synthesized by only two steps, and the preparation method is simple and has strong industrial applicability.

[0008] Based on the third aspect of the present application, the application of the aspirin derivative in preventing cardiovascular and cerebrovascular diseases, especially as a vasodilator, is also provided. By replacing aspirin with the aspirin derivative in the application of cardiovascular and cerebrovascular diseases, it is expected to reduce the side effects of long-term medication while maintaining the traditional therapeutic effect.

[0009] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0010] Firstly, an aspirin derivative has the following molecular structure expression:

[0011]

[0012] Secondly, a preparation method of the aspirin derivative comprises the following steps:

[0013]

[0014] S1, oxalyl chloride is added to an aspirin solution and stirred, dimethylformamide is added as a cosolvent, and the mixture is stirred at room temperature for 5-10 hours to generate 2-(acetyloxy)benzoyl chloride;

[0015] S2, saturated sodium bicarbonate solution is added to a histidine proline solution, the mixture is treated by ice bath, 2-(acetyloxy)benzoyl chloride is added, and the mixture is stirred for 10-24 hours; the obtained reaction solution is poured into hydrochloric acid solution, the precipitate is filtered, and the aspirin derivative is prepared by extraction and purification.

[0016] The histidine proline (H-HIS-PRO-OH) can be synthesized by any known solid-phase polypeptide synthesis method, liquid-phase polypeptide synthesis method, or recombinant DNA technology, enzyme method, etc., and is preferably synthesized by a solid-phase polypeptide method by a polypeptide synthesizer, for example, the following process: Fmoc / tBu solid-phase polypeptide synthesis method is used, then 20wt% hexahydropyridine and dimethylformamide are used to remove the protective group Fmoc, and the crude product is purified by column chromatography to obtain the product.

[0017] The molecular structure of the histidine proline (CAS: 20930-58-9) is expressed as follows, and can also be directly purchased or customized from existing suppliers for the purpose of saving production process:

[0018]

[0019] As a preferred scheme in the preparation method of the present application, in step S1, the molar ratio of aspirin to oxalyl chloride is 1:(1.5-4), such as 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, etc.

[0020] As a preferred scheme in the preparation method of the present application, in step S1, the solvent of the aspirin solution is one or more of dichloromethane, methanol, ethanol, diethyl ether, chloroform. Preferably, the addition amount of dimethylformamide cosolvent is 5-20 μL per gram of aspirin.

[0021] As a preferred scheme in the preparation method of the present application, after the reaction in step S1 is completed, the oil product is obtained by vacuum rectification, extracted with dichloromethane, and then distilled under reduced pressure again to collect the purified product.

[0022] As a preferred scheme in the preparation method of the present application, in step S2, the molar ratio of histidine proline to 2-(acetyloxy)benzoyl chloride is 1:(1-1.5), such as 1:1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, etc.

[0023] As a preferred scheme in the preparation method of the present application, in step S2, the molar ratio of histidine proline to sodium bicarbonate is 1:(2-5), such as 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, etc.

[0024] As a preferred scheme in the preparation method of the present application, in step S2, the solvent of the histidine proline solution is tetrahydrofuran.

[0025] As a preferred scheme in the preparation method of the present application, in step S2, the mass concentration of the hydrochloric acid solution is 5-15%; the amount of hydrochloric acid solution is sufficient to adjust the pH of the reaction solution to below 4, such as pH 3.9, 3.5, 3.0, 2.5, 2.0, 1.0, etc.

[0026] As a preferred scheme in the preparation method of the present application, in step S2, ethyl acetate and water are used for extraction, and after the extraction is completed, the organic layer is collected, the solvent is removed by vacuum distillation to obtain the product.

[0027] Again, the application of the aspirin derivative as described above and the aspirin derivative prepared by the method described above in preventing cardiovascular and cerebrovascular diseases, especially in the application as a vasodilator.

[0028] The present application can simply and efficiently prepare the aspirin derivative with both aspirin and histidine proline structure groups by a two-step synthesis process, which not only enhances the effect of aspirin in preventing and treating cardiovascular and cerebrovascular diseases, but also contains the histidine proline structure with low biological toxicity. It is accidentally found in the research process that the aspirin modified by the histidine proline can reduce drug side reactions, which has important significance for the development of new drugs. DETAILED DESCRIPTION

[0029] The present application will be further described by specific examples, and the examples described in the present application are only used to illustrate the present application and do not limit the scope of the present application.

[0030] In the following examples of the present application, the raw materials and reagents are purchased from commercially available products unless otherwise specified. Among them, histidine proline (H-HIS-PRO-OH) 95% is purchased from Nanjing Peptide Valley Biotechnology Co., Ltd.; acetylsalicylic acid (aspirin) 99% is purchased from Aladdin.

[0031]

Example 1

[0032] Acetylsalicylic acid (3.0 g, 16.65 mmol) was dissolved in 50 mL of dichloromethane and stirred at room temperature, and then oxalyl chloride (4.23 g, 33.3 mmol) was added and stirred for 30 min. Then 40 μL of dimethylformamide was added, and the reaction was stirred at room temperature for 8 h. The oil product was obtained by reduced pressure distillation, extracted by dichloromethane and distilled again under reduced pressure to collect the purified product 2-(acetyloxy)benzoyl chloride.

[0033] Histidine proline (312 mg, 1.24 mmol) was added to 6 mL of tetrahydrofuran, and then 2.5 mmol of saturated sodium bicarbonate solution was added, mixed and treated in an ice bath, and then 2-(acetyloxy)benzoyl chloride (283 mg, 1.49 mmol) was added and stirred for 15 h. After the reaction was completed, the obtained reaction solution was poured into 5% hydrochloric acid solution, and the precipitate was filtered, extracted and purified with ethyl acetate and water to prepare the aspirin derivative (350 mg, yield 66%).

[0034] 1H-NMR (300 MHz, DMSO-d6, δ ppm): 2.02 (m, 2H); 2.33 (m, 2H); 2.39 (s, 3H); 3.04 (m, 2H); 3.32 (m, IH); 3.51 (d, J = 3.2 Hz, 2H); 3.81 (d, J = 4.3 Hz, 2H); 3.95 (m, IH); 4.32 (m, IH); 7.26 (t, J = 3.5 Hz, IH); 7.37 (d, J = 3.7 Hz, IH); 7.66 (d, J = 6.4 Hz, IH); 7.84 (t, J = 4.3 Hz, IH); 7.81 (d, J = 5.4 Hz, IH); 8.73 (d, J = 5.7 Hz, IH); 12.22 (s, IH); 13.00 (s, IH).

[0035] Example 2

[0036] Acetylsalicylic acid (3.0 g, 16.65 mmol) was dissolved in 50 mL of dichloromethane and stirred at room temperature, and then oxalyl chloride (7.37 g, 58 mmol) was added and stirred for 30 min. Then 50 μL of dimethylformamide was added, and stirring was continued at room temperature for 10 h to complete the reaction. The oil product was obtained by distillation under reduced pressure, and then purified by extraction with dichloromethane and distillation under reduced pressure again to obtain the purified product, 2-(acetyloxy)benzoyl chloride.

[0037] Histidine proline (312 mg, 1.24 mmol) was added to 6 mL of tetrahydrofuran, and then 3.7 mmol of saturated sodium bicarbonate solution was added, and the mixture was ice-bathed, and then 2-(acetyloxy)benzoyl chloride (246 mg, 1.24 mmol) was added, and stirring was continued for 10 h to complete the reaction. After the reaction was completed, the obtained reaction solution was poured into 5% hydrochloric acid solution, and the precipitate was filtered to obtain aspirin derivative.

[0038] Example 3

[0039] Acetylsalicylic acid (3.0 g, 16.65 mmol) was dissolved in 50 mL of dichloromethane and stirred at room temperature, and then oxalyl chloride (3.2 g, 25 mmol) was added and stirred for 30 min. Then 20 μL of dimethylformamide was added, and stirring was continued at room temperature for 5 h to complete the reaction. The oil product was obtained by distillation under reduced pressure, and then purified by extraction with dichloromethane and distillation under reduced pressure again to obtain the purified product, 2-(acetyloxy)benzoyl chloride.

[0040] Histidine proline (312 mg, 1.24 mmol) was added to 6 mL of tetrahydrofuran, 4.5 mmol of saturated sodium bicarbonate solution was added, mixed and ice bathed, 2-(acetyloxy)benzoyl chloride (369 mg, 1.86 mmol) was added, and the reaction was stirred for 24 h. After the reaction was completed, the obtained reaction solution was poured into a 5% hydrochloric acid solution, and a precipitate was obtained by filtration, which was purified by extraction with ethyl acetate and water to obtain an aspirin derivative.

[0041] [Example 1] In vitro blood vessel ring experiment

[0042] (1) Preparation of Krebs solution: weigh NaCl 6.96 g, KCl 0.40 g, CaCl2 0.28 g, KH2PO4 0.062 g, MgSO4 0.144 g, NaHCO3 2.10 g, C6H 12 O6 2.10 g, and add distilled water to 1000 mL. Fill with a mixed gas of CO2 and O2 (volume ratio 5:95), adjust the pH of the solution to 7.4, and maintain the temperature at 37°C.

[0043] (2) Take SD rats, inject heparin anticoagulant intraperitoneally, and immediately take the thoracic aorta after painless dislocation under anesthesia to prepare a blood vessel ring with a length of 3-4 mm. The blood vessel ring is placed in a pre-prepared Krebs physiological bath. Adjust the basic tension of the blood vessel ring to 2 g, and change the solution every 15 min for 90 min.

[0044] (3) Vasodilation experiment

[0045] Add 0.6 mL of KCl solution with a concentration of 3 mol / L to a water bath with a volume of 30 mL (final concentration of KCl is 60 mmol / L) to stimulate the blood vessel ring to pre-contract. After the tension is balanced, gradually increase the concentration of the test drug (aspirin derivative prepared in Example 1) to produce a cumulative concentration relaxation curve, and observe the vasodilation response of different concentrations (10 -6 mol / L, 3×10 -6 mol / L, 10 - 5 mol / L, 3×10 -5 mol / L, 10 -4 mol / L) of aspirin derivative to KCl pre-contracted blood vessel ring, expressed as percentage of relaxation, i.e. percentage of pre-contracted degree. After each drug addition, the blood vessel tension reaches a plateau value before the next concentration is added. After one round of experiment, wash with 60 mmol / L KCl solution for three times, and then wash every 15 min until the blood vessel tension returns to the basic level before the experiment to start the next round of experiment.

[0046] (4) Vessel endothelium removal experiment

[0047] The two ends of the trimmed blood vessel ring were fixed, and a cotton swab with a diameter suitable for the inner diameter of the blood vessel was rubbed from the lumen twice in succession. After the blood vessel ring was hung stably for 1 h, 0.6 ml of KCl solution with a concentration of 3 mol / L was added to a water bath tank with a volume of 30 mL (the final concentration of KCl was 60 mmol / L), to stimulate the blood vessel ring to pre-contract, and after reaching the plateau value, whether the sample produced a relaxation response to 10 -6 mol / L acetylcholine was used as a standard for testing the presence of the blood vessel endothelium. When the relaxation amplitude was not more than 5% of the contraction amplitude, it was considered that the endothelium was completely removed, and the experiment could be started. After the tension was balanced, the concentration of the drug to be tested (the aspirin derivative prepared in Example 1) was gradually increased, and a cumulative concentration relaxation curve was drawn, to observe the relaxation response of different concentrations (10 -6 mol / L, 3×10 -6 mol / L, 10 - 5 mol / L, 3×10 -5 mol / L, 10 -4 mol / L) of the aspirin derivative to the KCl pre-contracted blood vessel ring, expressed by the percentage of relaxation, that is, the percentage of the relaxation degree to the pre-contraction degree. After each drug was added, the blood vessel tension reached the plateau value before the next concentration was added. After one round of experiment was completed, the blood vessel ring was washed with 60 mmol / L KCl solution for three times in succession, and then washed every 15 min, until the blood vessel ring tension returned to the baseline before the experiment, and then the next round of experiment could be started.

[0048] In this application example, the relaxation percentage test results of different concentrations of aspirin derivatives on the blood vessel ring are shown in Table 1:

[0049] Table 1, test results of isolated blood vessel ring experiment

[0050] Dosing concentrations <![CDATA[10 -6 mol / L]]> 3 x 10 -6 mol / L 10 -5 mol / L 3 x 10 -5 mol / L 10 -4 mol / L Vasodilation experiments 7.62±4.35％ 20.26±10.24％ 45.12±5.25％ 63.39±9.14％ 81.26±7.41％ Vessel denudation experiments 3.24±3.33％ 9.26±5.27％ 22.75±6.59％ 45.66±7.17％ 58.19±8.94％

[0051]

Application Example 2

[0052] Preparation of SD rats 16, 140-160g, 24 hours before modeling, fasting, free drinking water. SD rats were randomly divided into A, B two groups, each 8. A group of rats using 2% aspirin and 0.6mol / L hydrochloric acid mixed solution of equal volume ratio of gavage, a total of 3 times, each 13ml / kg, each interval 1.5 hours. B group of rats using 2% aspirin derivative prepared in example 1 and 0.6mol / L hydrochloric acid mixed solution of equal volume ratio of gavage, a total of 3 times, each 13ml / kg, each interval 1.5 hours. After the last gavage 4 hours, the rats were anesthetized and painlessly dislocated, quickly removed the whole stomach sample to observe the gastric mucosa tissue, and detect the serum prostaglandin PGE2 content (pg / ml) and plasma ET content of each group of rats, the results are shown in table 2.

[0053] Table 2, the results of erosive hemorrhagic gastritis experiment

[0054]

[0055] From the test results in table 2, the gastric mucosa tissue damage of A group of rats is significantly greater than that of B group, indicating that the aspirin derivative used in B group has lower damage to gastric mucosa than aspirin used in A group, which is beneficial to reduce drug side effects, etc.

[0056] The above only is the preferred embodiment of the present application, it should be pointed out that, for the ordinary skilled in the art, without departing from the method of the present application, can also make a number of improvements and supplements, these improvements and supplements should also be considered as the protection scope of the present application.

Claims

1. An aspirin derivative, characterized in that, The molecular structure expression is as follows: 。 2. A process for the preparation of an aspirin derivative according to claim 1, characterized in that, The method comprises the following steps: ； S1, oxalyl chloride is added into the aspirin solution and stirred, dimethylformamide is added as a cosolvent, the mixture is stirred at room temperature for 5-10 hours, and 2-(acetyloxy) benzoyl chloride is generated; S2, saturated sodium bicarbonate solution is added into the histidine proline solution, the mixture is treated by ice bath, 2-(acetyloxy) benzoyl chloride is added, and the mixture is stirred for 10-24 hours; the obtained reaction solution is poured into hydrochloric acid solution, the precipitate is obtained by filtration, and the aspirin derivative is obtained by extraction and purification.

3. The method for preparing an aspirin derivative according to claim 2, wherein: In step S1, the molar ratio of aspirin to oxalyl chloride is 1:(1.5-4).

4. The method for preparing an aspirin derivative according to claim 3, wherein: In step S1, the solvent of the aspirin solution is one or more of dichloromethane, methanol, ethanol, diethyl ether and chloroform.

5. Process for the preparation of an aspirin derivative according to any one of claims 2-4, characterized in that, After the reaction in step S1 is completed, the oil product is obtained by reduced pressure distillation, extracted by dichloromethane, and then distilled by reduced pressure again, and the purified product is collected.

6. The method for preparing an aspirin derivative according to claim 2, wherein: In step S2, the molar ratio of histidine proline to 2-(acetyloxy) benzoyl chloride is 1:(1-1.5).

7. The method for preparing an aspirin derivative according to claim 6, wherein: In step S2, the molar ratio of histidine proline to sodium bicarbonate is 1:(2-5).

8. The method for preparing an aspirin derivative according to claim 2, wherein: In step S2, the solvent of the histidine proline solution is tetrahydrofuran.

9. Process for the preparation of an aspirin derivative according to any one of claims 6-8, characterized in that, In step S2, the mass concentration of the hydrochloric acid solution is 5-15%; and the amount of the hydrochloric acid solution is used to ensure that the pH of the reaction solution is adjusted to be below 4.

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